APPLICANT'S ABSTRACT: The biological basis of ethanol intoxication and general anesthesia are believed to involve, in part, effects on ion channels that regulate nerve excitability. However, it is not known how ethanol and general anesthetics alter ion channel function at the molecular level and to what extent they share a common mechanism. The long-term goal of this proposal is to understand the molecular mechanism underlying inhibition of a cloned potassium channel by ethanol and other aliphatic alcohols (n-alcohols). Aliphatic alcohols behave as general anesthetics. The channel under study is encoded by Shaw2, a gene cloned from Drosophila. Previous studies in this laboratory have shown that Shaw2 potassium channels are selectively inhibited by clinically-relevant concentrations of ethanol in a manner consistent with a direct drug-channel interaction. Recombinant DNA technology, expression in frog oocytes or insect cells and patch-clamp recording will be used to address the following aspects: 1) The action of n-alcohols on the electrophysiological properties of Shaw2 potassium channels. 2)Identification of Shaw2 protein domains and amino acid residues that may interact with n-alcohols. 3)Overexpression, purification and reconstitution of recombinant Shaw2 potassium channels. The first two aspects will help us to understand the biophysical and molecular basis of channel inhibition. The third aspect will allow purification of sufficient quantities of the channel protein to permit the use of biochemical and biophysical methods to study more directly the interaction between n-alcohols and the channel. A comprehensive study of the molecular mechanism underlying inhibition of a potassium channel by n-alcohols is an important step towards understanding how ethanol can affect ion channel function in general, causing general anesthesia and other acute alterations affecting the brain and other organs.